Project Summary Metastasis is the leading cause of cancer-related deaths, accounting for 90% of all deaths. Intratumoral cellular heterogeneity contributes to metastasis and presents immense challenges to the development of effective targeted cancer therapeutics. Collective cell invasion, where adherent cells migrate as a single unit, is a major mode of metastasis. Our lab shows that cells within collective invasion packs display distinct phenotypic cellular heterogeneity and complex signaling pathways between pioneering ?leader? cells at the invasive front and their ?follower? cells behind. In order to further study these distinct cell types, we developed a novel technique termed Spatiotemporal Genomic and Cellular Analysis (SaGA) to select and isolate single leader or follower cells within collective invasion packs and then subject them to genomic or molecular biology approaches. Preliminary data using these isolated leader and follower cells shows that leader cells are a rare and highly invasive cell population that guide collective invasion packs, while highly proliferative but poorly invasive follower cells stream behind. Isolated leader cells and follower cells maintain their respective phenotypes over multiple passages, and genomic analysis shows differentially clustered gene expression between leader cells and follower cells. However, little is known about the functional significance of how leader cells and follower cells drive collective invasion, or how and why these distinct cell populations display mutually beneficial cell-cell cooperativity, i.e. symbiosis. Understanding how leader cell and follower cell cooperativity regulates collective cell invasion will provide crucial knowledge of mechanisms driving cancer metastasis. Therefore, we will test the central hypothesis that specialized leader cells and follower cells within a collective invasion pack promote mutually beneficial cell-cell symbiosis to provide a unique invasive advantage. To test this, we will determine 1) the molecular mechanisms that create cell-cell cooperativity between leader and follower cells and 2) how leaders and followers cooperate to promote tumor invasion and metastasis in vivo. In Aim 1, we will use 3-D spheroid invasion assays, live cell confocal imaging, and traditional cell biology methods to determine how angiogenic mimicry through VEGF regulates collective invasion and to identify how follower cells promote proliferation and survival of collective invasion packs. In addition, we will use liquid chromatography and mass spectrometry to characterize the leader-follower secretome and how these secreted proteins regulate collective invasion. In Aim 2, we will use a mouse orthotopic lung cancer model to assess how leader-follower cooperativity regulates tumor development, invasion, and metastasis in vivo. Additionally, we will use ex vivo imaging of precision cut lung slices to visualize and characterize leader-follower cooperativity during tumor collective invasion through the microenvironment. This work will ultimately allow us to elucidate novel signaling mechanisms that dictate cell-cell symbiosis during collective cancer invasion.